Vertebrate nitric oxide synthase (NOS) isoforms catalyze nitric oxide (NO) production for neuronal signalling, vasodilation, and destruction of pathogens, including liver-invading sporozoites of Plasmodium spp., the causative agents of malaria. Recent results from this laboratory indicate that Anopheles stephensi, a natural vector of Plasmodium, possesses a NOS gene (AsNOS) with striking homology to the recently described Drosophila NOS gene. Reverse transcriptase-polymerase chain reaction assays indicate that AsNOS is synthesized during P. falciparum and P. berghei infections, suggesting that anorpheline mosquitoes may possess an analogous NO killing mechanism for Plasmodium. Two objectives have been proposed for the characterization of AsNOS: (1) clone, sequence, and analyze enzyme activity of AsNOS, and (2) demonstrate NO toxicity to mosquito stage Plasmodium in vitro and in vivo. Sequence of full-length AsNOS and associated upstream regions will confirm the identity of AsNOS, provide insight into regulation of its expression, and facilitate construction of an expression system for enzyme analysis. Catalysis of NO production by AsNOS in vitro will confirm its behavior as a NOS isoform. Characterization of AsNOS in vitro will confirm its behavior as a NOS isoform. Characterization of AsNOS and its activity will provide new insights into mosquito-parasite biology, provide tools with which to examine NOS in other insect vectors, and perhaps provide a novel basis for engineering Plasmodium-refractory mosquitoes to interrupt the transmission of human malaria parasites.